Cells that are migrating in a chemical gradient display a polarized distribution of signaling and cytoskeletal components. We have investigated the mechanism of polarity establishment by using the model system Dictyostelium discoideum. We used both vegetative cells that are unpolarized and developed cells that have a distinct front and back and display a polarized morphology. In order to gain insight into the role of polarity during migration, we developed an open microfluidic device (OMD) that confines cells in narrow channels. The first version of the OMD was used to study the migration of unpolarized and polarized cells by luring them into microchannels with one micropipette generating a gradient of either folic acid (for vegetative cells) or cAMP (for developed cells). The second version used two micropipettes that were placed on either side of the microchannels and permitted gradient switching. We were able to observe the redistribution of signaling and cytoskeletal components as the cells reestablished their polarity after the gradient was switched. These experiments revealed that Ras activity and PTEN are reciprocally regulated, and suggest that high PI(4,5)P2 levels block Ras activity at the new leading edge. Similar findings were found in randomly moving and in dividing cells. F-actin polymerization occurs simultaneously with front extension, while microtubules redistribute to the rear and orient the microtubule organizing center (MTOC) and nucleus in polarized cells. Treated cells lacking an actin cytoskeleton had signaling responses, but did not re-localize the MTOC/nucleus in spatial gradients of chemoattractant. PTEN, which binds PI(4,5)P2 is not necessary for initial establishment of the cell rear but helps stabilize polarity by increasing backness at the rear of the cell.
Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07182013-092411 |
Date | 18 July 2013 |
Creators | Jowhar, Dawit Kamil |
Contributors | Dr. Charles Singleton, Dr. Ann Richmond, Dr. Andries Zijlstra, Dr. John Wikswo, Dr. Christopher Janetopoulos |
Publisher | VANDERBILT |
Source Sets | Vanderbilt University Theses |
Language | English |
Detected Language | English |
Type | text |
Format | application/pdf |
Source | http://etd.library.vanderbilt.edu/available/etd-07182013-092411/ |
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